Does the Current Fungicide Risk Assessment Provide Sufficient

Article pubs.acs.org/est

Does the Current Fungicide Risk Assessment Provide Sufficient Protection for Key Drivers in Aquatic Ecosystem Functioning? Jochen P. Zubrod,*,† Dominic Englert,† Alexander Feckler,†,‡ Natalia Koksharova,† Marco Konschak,† Rebecca Bundschuh,† Nadja Schnetzer,† Katja Englert,† Ralf Schulz,† and Mirco Bundschuh†,‡ †

Institute for Environmental Sciences, University of Koblenz-Landau, Fortstraße 7, D-76829 Landau, Germany Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Lennart Hjelms väg 9, SWE-75007 Uppsala, Sweden

‡

S Supporting Information *

ABSTRACT: The level of protection provided by the present environmental risk assessment (ERA) of fungicides in the European Union for fungi is unknown. Therefore, we assessed the structural and functional implications of five fungicides with different modes of action (azoxystrobin, carbendazim, cyprodinil, quinoxyfen, and tebuconazole) individually and in mixture on communities of aquatic hyphomycetes. This is a polyphyletic group of fungi containing key drivers in the breakdown of leaf litter, governing both microbial leaf decomposition and the palatability of leaves for leaf-shredding macroinvertebrates. All fungicides impaired leaf palatability to the leaf-shredder Gammarus fossarum and caused structural changes in fungal communities. In addition, all compounds except for quinoxyfen altered microbial leaf decomposition. Our results suggest that the European Union’s first-tier ERA provides sufficient protection for the tested fungicides, with the exception of tebuconazole and the mixture, while higher-tier ERA does not provide an adequate level of protection for fungicides in general. Therefore, our results show the need to incorporate aquatic fungi as well as their functions into ERA testing schemes to safeguard the integrity of aquatic ecosystems.

■

INTRODUCTION Fungal diseases are among the major threats for crop production,1 rendering the application of fungicides essential to secure the global food supply.2 Following their application, these substances can enter surface water bodies via point (e.g., wastewater treatment plant outlets) and diffuse (e.g., surface runoff) sources,3,4 potentially posing a risk to the integrity of aquatic ecosystems in general and their inhabiting fungi in particular. However, little is known about the sensitivity of nontarget fungal communities toward fungicides and, as a consequence, about the level of protection of the European Union’s environmental risk assessment (ERA) for these organisms and the ecosystem functions they provide.5,6 To assess this, aquatic hyphomycetes (i.e., a polyphyletic group of aquatic fungi), being of paramount importance for the breakdown of allochthonous organic matter (particularly leaf litter),7 seem to be a suitable starting point. In the process of organic matter breakdown, aquatic hyphomycetes accomplish two important functions: first, they can be the major drivers of biotic leaf mass loss and thus for the incorporation of the energy bound in leaves into stream food webs.7,8 Second, they transform leaves chemically and physically (i.e., conditioning), thereby increasing their palatability and nutritional value for leaf-shredding macroinvertebrates.9 However, there are no comprehensive studies targeting effects of individual synthetic © 2014 American Chemical Society

fungicides and their mixtures on both structural and functional end points in aquatic hyphomycetes (but see Zubrod et al.10 for inorganic fungicides), while several studies on single demethylation-inhibiting fungicides11−15 suggest low effect threshold concentrations. In this context, we empirically assessed whether current fungicide ERA practices in the European Union provide an acceptable level of protection for aquatic hyphomycetes using five current-use fungicides with different modes of toxic action (Table 1). These fungicides were investigated singly and in combination. The latter approach was prompted by the frequent detection of fungicides in complex mixtures in the field.4 Our experimental design allowed assessing the two important functions provided by aquatic hyphomycetes: i.e., microbial leaf decomposition as well as leaf palatability, which was studied via the food choice of the highly selective amphipod Gammarus fossarum KOCH,16 a key shredder in many European low order streams.17 Moreover, to gain a mechanistic understanding of potential implications at the functional level, the leaf-associated microbial communities were Received: Revised: Accepted: Published: 1173

October 16, 2014 December 16, 2014 December 17, 2014 December 17, 2014 DOI: 10.1021/es5050453 Environ. Sci. Technol. 2015, 49, 1173−1181

Article

Environmental Science & Technology Table 1. Products, Producers, Modes of Action, and Nominal Concentrations of the Fungicides experiment

product

producer

mode of action60

nominal concentrations (μg/L)

Azoxystrobin Carbendazim Cyprodinil Quinoxyfen Tebuconazole mixture

Ortiva Derosal Chorus Fortress 250 Folicur all of the above

Syngenta Agro Bayer CropScience Syngenta Agro Dow AgroSciences Bayer CropScience all of the above

inhibition of mitochondrial respiration inhibition of mitosis and cell division inhibition of amino acid and protein synthesis perturbation of signal transduction inhibition of sterol biosynthesis all of the above

20; 100; 500; 2500 5; 35; 245; 1715 8; 40; 200; 1000 5; 40; 320; 2560 1; 5; 50; 500 6; 60; 600; 3000

with leaves featuring microbial communities at different successional stages and thus presumably a high diversity of aquatic hyphomycetes.21 In the Hainbach near Frankweiler (Germany; 49°14′ N, 8°03′ E; upstream of any agricultural activity, settlement, and wastewater inlet), G. fossarum (cryptic lineage B22) were kicksampled 7 days prior to their use in experiments. Only adult males of approximately 6 to 8 mm body length being visually free from acanthocephalan parasites were used. During their acclimation (20 ± 1 °C, total darkness), gammarids were gradually adapted to amphipod medium (i.e., SAM-5S;23 for composition see Table S3). For the first 3 days in the laboratory, amphipods received preconditioned black alder leaves ad libitum. Afterward, they were starved to level their appetite. Main Experiments on Functional and Structural Effects. Each experiment comprised four fungicide concentrations plus a fungicide-free control. To assess both functional end points (i.e., microbial leaf decomposition and gammarids’ food choice), sets of four leaf discs (diameter = 16 mm) were cut out of unconditioned leaves. The discs were dried at 60 °C for 24 h and weighed individually to the nearest 0.01 mg. Subsequently, two discs per leaf were conditioned in the fungicide-free control, while the remaining two were exposed to one of the fungicide concentrations. For this purpose, after sewing them individually into labeled and thoroughly leached fiber glass gaze bands, the discs were placed in 5-L round glass aquaria (seven for each of the five fungicide concentrations) together with additional discs for the assessment of structural end points (see below). Each aquarium was filled with 4 L of conditioning medium amended with the respective fungicide concentration(s) and received 10 g (wet weight after blotting) of inoculum. Aquaria were kept at 16 ± 1 °C in total darkness and were continuously stirred and aerated. Test solutions were renewed every 3 days to ensure a continuous fungicide exposure over the 12-day conditioning period. This chronic exposure without preconditioning of leaf discs in fungicide-free medium was considered the realistic worst-case scenario given the continuous presence of fungicides in agricultural streams.24 After 12 days, the leaf discs intended for assessing the functional end points were rinsed in fungicide-free SAM-5S for 30 min. Two leaf discs originating from the same leaf (i.e., one conditioned in the control and one conditioned in the presence of fungicide(s)) were fixed in the center of a food-choice arena (= 300 mL crystallization dish) filled with 100 mL fungicidefree SAM-5S.25 Subsequently, one gammarid was allowed to feed on the two discs for 24 h (at 20 ± 1 °C in total darkness). In the same arena, the two corresponding discs from the same leaf were protected from amphipod feeding using a 0.5 mm fiberglass mesh screen. These discs were used to quantify the microbial leaf decomposition and abiotic mass losses during the experiments. Additionally, this procedure allowed for controlling for these factors when calculating gammarids’ leaf

characterized. We expected the fungicides to have negative effects on both aquatic hyphomycete community structure and functioning,11−15 while their mixture effect was hypothesized to meet or exceed reference model predictions as shown for the direct toxicity (i.e., exposure via water phase) of the same fungicide mixture toward G. fossarum.18

■

MATERIAL AND METHODS Chemicals. For the five single-substance experiments, nominal concentrations of the tested fungicide active ingredients (applied as products; Table 1) were selected considering effect concentrations reported for aquatic hyphomycetes and other aquatic fungi,11−15,19 aiming to observe a gradient from weak to strong effects on leaf-associated fungal communities’ structure and functioning. The mixture experiment employed a fixed-concentration ratio with the total fungicide concentrations covering approximately those of the single-substance experiments (i.e., 1, 10, 100, and 500 μg/L for each substance; Tables 1 and S1). The only exception was quinoxyfen, which was applied at concentrations twice as high as the remaining substances (i.e., 2, 20, 200, and 1000 μg/L; Tables 1 and S1) to counteract its fast dissipation from the water phase (Table S2). For the preparation of stock solutions and subsequent serial dilution, a nutrient medium (i.e., conditioning medium; for composition see Table S3) was used.20 Shortly after fungicide application (i.e., after thorough mixing but before introduction of leaf material) and at the time of the first medium renewal (i.e., after 3 days; see below), triplicate 10 mL samples of the treatments with the lowest and the highest tested concentrations as well as one sample from the respective controls were frozen (−20 °C) until further processing. Fungicide concentrations were verified using ultrahigh performance liquid chromatography−mass spectrometry (Thermo Fisher Scientific).13 Since measured initial concentrations and nominal ones agreed well (Table S2), the latter are reported throughout this paper. Unless otherwise specified, chemicals were purchased from Sigma-Aldrich or Roth. Sources of Leaves, Microorganisms, and Gammarids. Experimental procedures are described in more detail in Zubrod et al.10 Briefly, Alnus glutinosa (L.) GAERTN. (black alder) leaves were collected in October 2011 (near Landau, Germany; 49°11′ N, 8°05′ E) and stored at −20 °C until further use. For each of the six experiments, 500 alder leaves were deployed in fine-mesh bags (0.5 mm mesh size; 10 leaves per bag) in the Rodenbach near Grünstadt (Germany; 49°33′ N, 8°02′ E) upstream of any agricultural activity, settlement, and wastewater inlet for 14 days. Back in the laboratory, another 500 leaves were added and leaves were mixed and kept for a further 14 days in 30 L of aerated conditioning medium at 16 ± 1 °C in total darkness (complete medium renewal after 7 days) before being used as microbial inoculum in the experiments (see below). This procedure ensured inoculum 1174

DOI: 10.1021/es5050453 Environ. Sci. Technol. 2015, 49, 1173−1181

Article

Environmental Science & Technology

Figure 1. Mean or median (with 95% CI) percentage reductions (compared to the respective control) in microbial decomposition of leaf material conditioned in the presence of different concentrations of (A) azoxystrobin, (B) carbendazim, (C) cyprodinil, (D) quinoxyfen, (E) tebuconazole, and (F) the fungicide mixture. Asterisks denote statistically significant differences compared to the respective control.

medium was renewed four times) that potentially adsorbed to the leaf discs’ surfaces. After 3 days, leaf discs from the control and from the fungicide treatment were rinsed and simultaneously offered to one gammarid for 24 h (n = 49). Characterization of Leaf-Associated Microbial Communities. Microbial communities were characterized on leaf discs conditioned together with the discs used for assessing the functional end points during the main experiments resulting in seven independent replicates per treatment. Aquatic hyphomycete community composition was assessed via spore morphology generally following Bärlocher.29 Leaf discs were agitated in deionized water for 96 h at 20 °C before fixing and staining fungal spores by adding lactophenol cotton blue. Spores were identified under a microscope using several identification keys (e.g., Ingold30). Ergosterol was quantified as a proxy for leaf-associated fungal biomass according to Gessner.31 After extraction in alkaline methanol, ergosterol was purified by solid-phase extraction (Sep-Pak Vac RC tC18 500 mg sorbent, Waters) and quantified by high-performance liquid chromatography (1200 Series, Agilent Technologies). Since leaf-associated bacteria can interact with aquatic hyphomycetes,32 bacterial densities (number of cells per mg leaf) were determined according to Buesing.33 Dislodged

consumption. After 24 h, the gammarids (49 per fungicide concentration) as well as any remaining leaf material were dried and weighed as described above. Replicates containing animals that died or molted during the experiments were excluded from evaluation of the food-choice data. Supplemental Study on the Repellent Effect of Fungicides. To assess the potential impact of a repellent effect caused by adsorbed fungicides (cf. Hahn and Schulz26 and Rasmussen et al.27), additional experiments were performed that excluded any fungicide-induced changes in the leaf-associated microbial communities. Therefore, preconditioned leaf discs28 were weighed, autoclaved, and exposed to each of the individual fungicides and the mixture in 4 L of autoclaved conditioning medium containing 10 g (wet weight after blotting) of autoclaved microbial inoculum simulating the procedure detailed above. While one aquarium contained no fungicides (i.e., control), the second one contained the respective fungicide or the mixture at an aqueous-phase concentration 4-times higher than the lowest concentration triggering a significant food-choice response in the main experiments (or 4-fold the highest concentration if no significant effect was found). This procedure was selected to account for the total amount of fungicide applied during microbial conditioning in the main experiments (note that the 1175


Article


Figure 2. Mean or median (with 95% CI) fungal biomass (circles) and bacterial density (triangles) relative to the respective control associated with leaf material conditioned in the presence of different concentrations of (A) azoxystrobin, (B) carbendazim, (C) cyprodinil, (D) quinoxyfen, (E) tebuconazole, and (F) the fungicide mixture. Asterisks denote statistically significant differences to the respective control.

Univariate data were checked for normality by visual inspection, while homoscedasticity was tested via Levene’s test. To assess statistically significant differences between the fungicide treatments and the corresponding controls for microbial leaf decomposition and gammarids’ leaf consumption, paired t-tests or, as a nonparametric alternative, Wilcoxon signed-rank tests were used (note the paired design according to the criteria for instance described by Zar34). For the remaining univariate data (for instance fungal biomass and bacterial density), fungicide treatments were compared to the controls by performing ANOVAs followed by Dunnett’s tests orif the assumptions for parametric testing were not met Kruskal−Wallis tests followed by Wilcoxon rank-sum tests using the Bonferroni adjustment for multiple comparisons (for all univariate analyses see Zar34). To assess shifts in aquatic hyphomycete community composition, permutational multivariate analysis of variance (PERMANOVA) was used, which is a nonparametric, permutation-based procedure to assess for statistically significant differences between groups of multivariate data.35 Analyses were performed on Bray−Curtis dissimilarities, while data were square-root transformed to reduce the effect of dominant

bacteria were stained using SYBRGreen II (Molecular Probes). Subsequently, digital pictures were taken using an epifluorescence microscope and cells were counted by image analysis software (Axio Scope.A1, AxioCam MRm, and AxioVision, Carl Zeiss MicroImaging). Statistical Analyses. Microbial leaf decomposition (D) was calculated as D = (nb0.74 − na)/t.10 There, nb and na refer to the initial and final dry weight of the leaf discs protected from amphipod feeding, respectively, 0.74 is an empirical factor controlling for leaching of the leaf material, and t is decomposition time (i.e., 13 days). Gammarids’ leaf consumption (C) was calculated as C = [(f b − fa) − (nb − na)]/ (gt) and C = ( f bk − fa)/(gt) for the main and supplemental food-choice experiments, respectively.25,28 There, f b is the initial dry weight of the leaf disc accessible for the gammarid and fa is the dry weight of the same disc after the food-choice experiment. The gammarid’s dry weight is indicated by g, and the feeding time (t) was 1 day. The leaf weight correction factor k, correcting for abiotic mass losses during the supplementary experiments, was calculated experiment-wise as k = ∑(1 − (nb − na)/nb)/10 using the initial (nb) and final (na) dry weight of 10 leaf discs not accessible for gammarids. 1176


Article


Figure 3. Mean or median percentage difference (with 95% CI) in the feeding of G. fossarum on leaf discs conditioned in the presence of different concentrations of (A) azoxystrobin, (B) carbendazim, (C) cyprodinil, (D) quinoxyfen, (E) tebuconazole, and (F) the fungicide mixture compared to control discs. Positive effect sizes imply less feeding on the fungicide-exposed discs compared to the control and vice versa. Asterisks denote statistically significant differences compared to control leaf discs.

azoxystrobin (at 100 and 500 μg/L; Figure 1A; group means or medians on the original scale as well as all P values are provided in Table S4), carbendazim (at ≥245 μg/L; Figure 1B), and cyprodinil (at ≥200 μg/L; Figure 1C), while the observed effect sizes cannot be explained by the mass of fungicides potentially adsorbed to the leaf discs (Table S6). Since aquatic hyphomycete speciesthe main drivers of microbial decomposition7differ in their leaf decomposition efficiency,40 fungicide-induced changes in fungal community composition may be the reason for this functional response. Accordingly, fungal communities exposed to these three substances separated well from the respective controls, with statistically significant differences in their composition (judged with PERMANOVA) being observed for azoxystrobin (at ≥20 μg/ L) and carbendazim (at ≥35 μg/L). For cyprodinil, statistically nonsignificant but considerable shifts in the fungal community composition were found at concentrations ≥40 μg/L (Figures S1 and S2), being corroborated by a significantly reduced number of leaf-associated fungal species (at ≥40 μg/L; Table S8). In contrast, quinoxyfen and tebuconazole did not negatively affect microbial leaf decomposition (Figure 1D,E). However, 1 μg of tebuconazole/L unexpectedly resulted in a significantly

species.35 Species determined in only one sample of an experiment were excluded to reduce arbitrary noise. In the case of samples without fungal spores, which was mostly related to fungicide treatment, a “dummy species” with an abundance of one was added to each sample of the respective experiment since Bray−Curtis dissimilarity is undefined for empty samples.36 Results of additional analyses and visualizations of the aquatic hyphomycete communities are provided in the Supporting Information. To assess the joint effects of the five fungicides during the mixture experiment, observed effect sizes were compared to predictions by a reference model for mixtures composed of components with dissimilar modes of toxic action, namely “independent action,”37 whose applicability on community level effects was already shown.38 More information about the mixture predictions can be found in the Supporting Information. For statistics and figures, R version 3.0.2 together with the add-on packages “drc,” “multcomp,” “plotrix,” and “vegan” was used.39

■

RESULTS AND DISCUSSION Microbial Leaf Decomposition. As hypothesized, microbial leaf decomposition was significantly negatively affected by 1177


Article


Table 2. Toxicity End Points (Extracted from the Pesticide Properties DataBase55) from Standard Toxicity Tests with the Resulting Regulatory Acceptable Concentrations (RAC; End Points Used for Calculation Are Printed in Bold) for the Assessed Substancesa acute standard tests

chronic standard tests

fungicide

Daphnia

fish

algae

Azoxystrobin Carbendazim Cyprodinil Quinoxyfen Tebuconazole

230 150 220 80 2790

470 190 2410 270 4400

360 >7700 2600 27 1960

Chironomus 800 13.3 240 128 2510

Daphnia 44 1.5 8.8 28 10

fish 147 3.2 83 14 12

RAC

NOEC

end point(s)b

2.3 0.15 0.88 0.8 1

Does the Current Fungicide Risk Assessment Provide Sufficient

Recommend Documents